Xenotransplantation presents an effective solution to donor organ shortage but vigorous immune response across specie barrier limits its success. The level of current immunosuppression used to prolong xenograft survival is lethal and also impractical for clinical use. Therefore, there is a strong need to develop methods to limit immunosuppression and induce immunological tolerance or accommodation. Recently characterized CD4+CD25+ T regulatory cells (Tregs) offer some hope of inducing donor specific immune modulation without much lethality. One of the limitations of their therapeutic use is their presence in very low numbers. In this study we have tried to isolate and expand baboon Tregs and measured their effectiveness in suppressing pig to baboon xenogeneic response.[unreadable] Tregs were isolated from peripheral blood, spleen and lymph nodes of healthy baboons first by CD4+ cells enrichment by magnetic beads and then by sorting CD4+ CD25+ cells via FACS. These sorted cells were tested for their suppressive potential by addition to the co culture of irradiated pig PBMCs and CD4+ CD25- cells. The isolated Tregs were also expanded in culture for 4 weeks in presence of IL2 and irradiated pig PBMCs and were evaluated for their inhibitory effect. Further, flow cytometric assays for intracellular cytokines and surface expression of various activation markers were also performed to study the mechanism of action of these Tregs in this xenotransplant model.[unreadable] The Treg isolation technique was very effective and 97% pure Tregs (1-2% of CD4+ T cells) were isolated. These isolated cells effectively suppressed the vigorous CD4+CD25- cell proliferative response to irradiated pig PBMCs (85 % suppression). These cells also expressed high levels of FoxP3, a potent marker of Tregs. Isolated Tregs expanded 150-200 folds in culture and were also able to suppress CD4+CD25- cells in a similar manner as naove Tregs. Tregs also suppressed the cytokine production by the CD25- cells in response to pig PBMCs, suggesting a possible mechanism of Treg function.[unreadable] With the above experiments it is clear that Tregs are potent suppressors of pig to baboon xenogenic response and they can also be expanded in vitro to achieve sufficient quantity without losing their suppressive potential. Thus, Tregs offer a potential non lethal alternative to non specific immunosuppression currently used to overcome xenograft rejection.[unreadable] In addition to these cellular studies we have commenced performance of pig to baboon orthotopic cardiac xenotransplantation. As a result we are verifying the stability of this animal model and establishing a set of baseline control results from which to compare additional transplants treated with pharmacologic and cell based immunosuppression.